Hepatitis C virus (HCV) is a major cause of community-acquired viral hepatitis. Infected individuals are at increased risk of developing chronic liver disease and HCV sequelae are now the most common indication for liver transplantation in the US. We demonstrated that HCV could be classified into 6 major genotypes and numerous subtypes. Prototype strains of the various genotypes of HCV have been biologically amplified in chimpanzees, packaged and distributed for use as challenge inocula in studies of passive and active immunoprophylaxis, etc. Full-length cDNA clones of HCV [genotypes 1a (strains H77 and HC-TN), 1b (strains HC-J4 and Con1) and 2a (strain HC-J6)] have been constructed and transcribed RNA used to transmit hepatitis C to chimpanzees by in vivo hepatic transfection. With the reagents we have developed we are pursuing a collaborative study of the immunopathogenesis of HCV infections in chimpanzees, a surrogate of man. We have shown that humoral immunity is apparently not important in the control of acute infection or in preventing reinfection. Antibodies against the viral envelope proteins develop in animals that become chronically infected, but not in animals with resolved infections. Furthermore, neutralizing antibodies, as measured against pseudotyped retroviruses bearing the HCV glycoproteins, was only detected in animals that became chronically infected. Intrahepatic cellular immune responses are vigorous in chimpanzees with viral control but their strength does not necessarily predict the final outcome of infection. Also, gene expression analysis of acutely infected chimpanzees with different outcomes identified intrahepatic responses associated with HCV viremia, such as interferon alpha, as well as outcome specific responses associated with clearance, such as genes involved in antigen processing and presentation, the adaptive immune response and interferon gamma induction. We have also studied the correlation between host response, virus evolution and outcome in chimpanzees infected with a unique HCV strain associated with severe acute liver disease. Analysis of the entire polyprotein sequence of viruses recovered during the first year of follow-up suggested strong positive selection of variant sequences. Thus, strong host cellular immune responses are closely related to the emergence of new virus variants. However, the emergence of such variants does not necessarily lead to viral persistence. Since animals infected with the same HCV strain had mild to severe acute liver disease these studies also suggested that the host response is the principal determinant of the severity of acute HCV. We have demonstrated that sterilizing immunity can be achieved by repeated infection of chimpanzees, but that this sterilizing immunity is strain-specific. We have demonstrated that the observed immunity is not mediated by neutralizing antibodies, but is correlated with anamnestic T cell responses. We have also demonstrated that immunity acquired during acute HCV infection does not necessarily prevent persistent infection, even following rechallenge with the homologous monoclonal virus. Preliminary analyses suggest that the persisting viruses represent immune escape variants. These studies will provide an in-depth analysis of humoral versus cellular immune responses to HCV infection, and their association with virus evolution. The availability of infectious cDNA clones of HCV has permitted a mutational analysis of genomic regions. For example, individual portions of the 3' UTR have been deleted from the full-length genotype 1a clone and the resultant deletion mutant clones inoculated into chimpanzees by intrahapatic transfection. Several regions of the UTR have been identified as critical for in vivo replication of HCV. In other studies we have deleted the hypervariable region 1 (HVR1) of the E2 protein of HCV, the region that contains a neutralization epitope. Surprisingly, the deletion mutant virus was viable but attenuated when transfected into chimpanzees. Adaptive mutations that increased the fitness of this deletion mutant were identified. This study also indicated that HVR1 is not essential for the resolution of infection or the progression to chronicity since both outcomes were observed with HCV lacking HVR1. Most recently, we have performed a mutational analysis of the gene that encodes the p7 protein, a protein without a well-defined function. By testing deletion mutants and mutants with point mutations we have demonstrated that p7 is critical for the viability of HCV. By testing chimeras between our genotype 1a and 2a clones we have shown also that p7 contains critical genotype-specific sequences located within the amino- and/or carboxy- terminal intraluminal tails of p7. In a collaborative study we have extended the mutagenesis studies to determine the in vivo effect of mutations that permit replication of a subgenomic replicon derived from the HCV strain Con1 in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes increased significantly by introducing three adaptive mutations in NS3 and NS5A. However, these cell culture-adaptive mutations negatively influenced in vivo infectivity. Thus, mutations that are adaptive for replication of HCV in cell culture might be highly attenuating in vivo. Since most, if not all, replicons studied contain adaptive mutations specific for Huh-7 cells, our study has important implications for the interpretation of the biological relevance of findings in this in vitro system. In other studies, we have constructed chimeric genomes from infectious cDNA clones of HCV and bovine viral diarrhea virus. These genomes can replicate in transfected cells but the resultant viral products cannot assemble into infectious virus in the absence of helper virus. However, the transfected genome expresses large quantities of structural HCV proteins in susceptible cells. We have expressed modified HCV envelope glycoproteins in vitro and in vivo and tested their potential immunogenecity. A DNA vaccine expressing a truncated E2 protein generated high anti-E2 titers, including anti- HVR1, in chimpanzees, but it did not prevent infection following homologous monoclonal challenge. It might however have influenced the outcome of the infection since both vaccinated chimpanzees had early clearance of viremia. Studies are being performed to determine whether the expressed envelope proteins generated neutralizing antibodies in vivo. We have constructed an infectious cDNA clone of GB virus-B (GBV-B), a monkey virus that is the closest relative to HCV. Experimental infection with GBV-B results in acute viral hepatitis in tamarins. In addition, we have prepared challenge pools of GBV-B and determined the infectivity titer of these in tamarins. We have shown that GBV-B can infect owl monkeys, but not chimpanzees, suggesting that this virus is not of human origin. We are currently using the GBV-B tamarin system to study characteristics of the virus that it shares with HCV. We have demonstrated that sterilizing immunity to GBV-B can be achieved by repeated infection of tamarins and that this sterilizing immunity was not due to a high titer of neutralizing antibodies. By testing deletion mutants of the GBV-B clone in tamarins we have shown that each of the predicted encoded GBV-B proteins is critical for the virus. However, viruses with deletions of specific domains within the 3' UTR were found to be viable. Interestingly, infection with one such deletion mutant produced a persistent infection with chronic hepatitis. This observation strengthens the relevance of GBV-B as a surrogate model for the study of HCV. Most recently, we have demonstrated that a p7 like protein of GBV-B exists and that this protein most likely is twice as large as the equivalent protein of HCV.